Project Summary/Abstract Listeria monoctyogenes vector-based vaccines have shown considerable promise in preclinical studies and in human therapeutic vaccine trials as well. Indeed, there are many features that make L. monocytogenes attractive, however, there has never been a systematic attempt to optimize its interactions with the host innate immune system to produce more potent and versatile vaccines. During the past few years, there has been considerable progress defining the host pathways of innate immunity that result from infection. Work in our laboratory has identified listerial strains that activate enhanced and/or diminished innate immune responses. For example, strains that overexpress multidrug efflux pumps (MDRs), due to a mutation in the MDR repressors (TetR), induce 20-times more IFN&#946;, while strains deleted for another MDR (MdrM) induce 3-fold less. We identified c-di-AMP as the L. monocytogenes secreted ligand that activates this response. We also identified strains that trigger two separate pathways that lead to inflammasome activation, cell death and the secretion of 10-times more IL-1&#946;. One strain has been engineered to lyse in the macrophage cytosol and induces a DNA-dependent, AIM2-dependent inflammasome, while another strain secretes an ActA-flagellin (ActA-Fla) fusion protein and activates a NLRC4-dependent inflammasome. The overall goal of this proposal is to evaluate potential vaccine strains that differ markedly in the induction of these pathways. The overall hypothesis is that by inducing altered levels of innate immune pathways, we will identify strains and pathways that lead to longer-lived, more potent vaccines. In Aim I, a panel of strains will be constructed that induce altered levels of innate immune pathways. Strains with a mutation in MdrM and the c-di-AMP cyclase are predicted to induce very little, if any, IFN&#946;. Strains lacking TetR and the c-di-AMP phosphodiesterase are predicted to induce very high levels of IFN&#946;. Strains that lyse in the cytosol or express ActA-Fla will be used to explore the role of inflammasome activation. In Aim 2, the cell biology of infection will be examined, both for bacterial intracellular growth and for host cell cytotoxicity. Infected macrophages will be characterized for activation of both transcriptional and post-transcriptional responses. Macrophages from knockout mice bred in our mouse core will be used to test our hypothesize about the precise pathways involved. In Aim 3, the strains will be characterized in mouse models. Immunization dose and time to challenge will be varied to examine the strains for their capacity to induce long-lived immunity. Strains engineered to express ovalbumin and four separate vaccinia CD8+ T-cell epitopes will be used to gauge the strength and duration of CD8 T-cells specific to foreign antigens. Lastly, the capacity of the new strains to function as potent vaccines will be examined in a vaccinia model of immunity and in a collaboration, to examine protection in a mouse model of malaria. If successful, these studies will lead to the development of a new generation of L. monocytogenes-based vaccines for clinical use.